CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

54 Pags.- 19 Figs.- 2 Tabls. The definitive version is available at: https://nph.onlinelibrary.wiley.com/journal/14698137

Bibliographic Details
Authors: Wang, Longlong, Rubio Luna, María Carmen, Xin, Xian, Zhang, Baoli, Fan, Qiuling, Wang, Qiang, Ning, Guogui, Becana Ausejo, Manuel, Duanmu, Deqiang
Format: article
Status:Versión aceptada para publicación
Publication Date:2019
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:dnet:digitalcsic_::c197693f2877407c7b8fb2ff4ad6777c
Online Access:http://hdl.handle.net/10261/191637
Access Level:Open access
Keyword:CRISPR/Cas9
leghemoglobins
nitrogen fixation
nodule senescence
phytoglobins
reactive oxygen species
RNA-seq.
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spelling CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixationWang, LonglongRubio Luna, María CarmenXin, XianZhang, BaoliFan, QiulingWang, QiangNing, GuoguiBecana Ausejo, ManuelDuanmu, DeqiangCRISPR/Cas9leghemoglobinsnitrogen fixationnodule senescencephytoglobinsreactive oxygen speciesRNA-seq.54 Pags.- 19 Figs.- 2 Tabls. The definitive version is available at: https://nph.onlinelibrary.wiley.com/journal/14698137Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA‐sequencing (RNA‐seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly‐β‐hydroxybutyrate and disruption of mitochondria. RNA‐seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb‐deficient nodules had c. 30–50‐fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post‐translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.This work was supported by grants from the National Natural Science Foundation of China (31870220 and 31570233), the Fundamental Research Funds for the Central Universities (Program No. 2662015PY171), and Spanish Ministry of Economy and Competitiveness‐European Regional Development Fund (AGL2017‐85775‐R).Peer reviewedJohn Wiley & SonsNational Natural Science Foundation of ChinaFundamental Research Funds for the Central Universities (China)Ministerio de Economía y Competitividad (España)Rubio Luna, María Carmen [0000-0002-9378-7775]Becana Ausejo, Manuel [0000-0002-1083-0804]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201920192019info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionhttp://hdl.handle.net/10261/191637reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/AGL2017-85775-Rhttps://doi.org/10.1111/nph.16077Síinfo:eu-repo/semantics/openAccessoai:dnet:digitalcsic_::c197693f2877407c7b8fb2ff4ad6777c2026-05-22T06:33:51Z
dc.title.none.fl_str_mv CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
title CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
spellingShingle CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
Wang, Longlong
CRISPR/Cas9
leghemoglobins
nitrogen fixation
nodule senescence
phytoglobins
reactive oxygen species
RNA-seq.
title_short CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
title_full CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
title_fullStr CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
title_full_unstemmed CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
title_sort CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation
dc.creator.none.fl_str_mv Wang, Longlong
Rubio Luna, María Carmen
Xin, Xian
Zhang, Baoli
Fan, Qiuling
Wang, Qiang
Ning, Guogui
Becana Ausejo, Manuel
Duanmu, Deqiang
author Wang, Longlong
author_facet Wang, Longlong
Rubio Luna, María Carmen
Xin, Xian
Zhang, Baoli
Fan, Qiuling
Wang, Qiang
Ning, Guogui
Becana Ausejo, Manuel
Duanmu, Deqiang
author_role author
author2 Rubio Luna, María Carmen
Xin, Xian
Zhang, Baoli
Fan, Qiuling
Wang, Qiang
Ning, Guogui
Becana Ausejo, Manuel
Duanmu, Deqiang
author2_role author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv National Natural Science Foundation of China
Fundamental Research Funds for the Central Universities (China)
Ministerio de Economía y Competitividad (España)
Rubio Luna, María Carmen [0000-0002-9378-7775]
Becana Ausejo, Manuel [0000-0002-1083-0804]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv CRISPR/Cas9
leghemoglobins
nitrogen fixation
nodule senescence
phytoglobins
reactive oxygen species
RNA-seq.
topic CRISPR/Cas9
leghemoglobins
nitrogen fixation
nodule senescence
phytoglobins
reactive oxygen species
RNA-seq.
description 54 Pags.- 19 Figs.- 2 Tabls. The definitive version is available at: https://nph.onlinelibrary.wiley.com/journal/14698137
publishDate 2019
dc.date.none.fl_str_mv 2019
2019
2019
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/191637
url http://hdl.handle.net/10261/191637
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/AGL2017-85775-R
https://doi.org/10.1111/nph.16077
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv John Wiley & Sons
publisher.none.fl_str_mv John Wiley & Sons
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,81155

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